Abstract Control of gene expression in eukaryotic organisms has been extensively studied in a broad range of experimental systems, including the apicomplexan parasites responsible for several human diseases. Changes in the three-dimensional (3D) organization of chromosomes within the nucleus has been associated with changes in gene expression in model organisms, although whether alterations in subnuclear chromosome structure are causative or simply associated with transcriptional changes is a matter of debate. Similar phenomena have been reported by a number of groups studying Plasmodium, the genera of parasites responsible for malaria in both humans and animals. For example, perinuclear clusters of the subtelomeric chromosomal domains have been observed in P. falciparum and linked to control of expression of variant antigen encoding genes. However, definitive evidence defining the role of 3D arrangement in regulating gene expression, including gene involved in antigenic variation or virulence factors in eukaryotic pathogens are limited. Here we propose to explore the hypothesis that 3D, higher-order chromosomal organization evolved in parasites of primates to help coordinate monoallelic variant gene expression. We recently performed comparative studies on subnuclear chromosomal organization in different species of malaria parasites and found that parasites that infect primates display a unique, highly complex organization not observed in malaria parasites of rodents. We have also recently created several genetically modified lines of P. falciparum that have significant changes in specific elements proposed to be involved in chromosomal organization. Several of these modifications have profound effects on expression of genes involved in antigenic variation. We will use these resources to investigate how epigenetics features and chromosome organization are maintained in apicomplexan parasites as well as their role in coordinating expression of large, multicopy gene families, such as var genes. We will perform the following two specific aims. In AIM 1, we will characterize the evolution of 3D chromatin structure in apicomplexan parasites by performing Chromosome Conformation Capture (Hi-C) experiments to analyze the spatial organization of chromosomes for several parasites of the apicomplexan clade. We will also use genetically altered parasite strains to identify DNA sequence elements involved in 3D chromosomal organization. In AIM 2, we will investigate a possible link between 3D higher-order chromosomal organization and monoallelic variant antigen expression in primate Plasmodium species by analyzing genetically altered parasites with deletions of regulatory elements that alter monoallelic variant antigen (var) expression. The results of this work will be transformative for our understanding of chromatin structure's role in parasite gene expression, virulence and disease. Our main goal is to design novel intervention strategy targeting chromatin elements that can interrupt the development and immune evasion strategies of malaria parasites. !